The present invention relates in general to the control of cellular and neural activity and to the treatment of cellular and neural disorders. More particularly, the present invention is directed to methods and associated compositions and medicaments for the modification of mammalian cellular and neural activity through the administration of carbon monoxide dependent guanylyl cyclase modulating purine derivatives which selectively and controllably induce the in vivo genetic expression of naturally occurring genetically encoded molecules including neurotrophic factors. The methods, compositions, and medicaments of the present invention may be used to affect a variety of cellular and neurological activities and to therapeutically or prophylactically treat a wide variety of physiological, neurodegenerative, and neurological disorders.
The evolution of the central nervous system in mammals was a natural response to an increasingly complex environment requiring solutions to difficult problems. The resulting structure is an intricate biochemical matrix that is precisely controlled and attenuated through an elaborate system of chemically modulated regulatory pathways. Through an elaborate series of highly specific chemical reactions, these pathways oversee and direct every structural and operational aspect of the central nervous system and, through it, the organism itself. Normally the complex interplay of the various control systems cooperates to produce a highly efficient, versatile central nervous system managed by the brain. Unfortunately, when the biochemical matrix of the central nervous system is damaged, either through age, disease or other reasons, the normal regulatory pathways may be incapable of effectively compensating for the loss. In such cases it would be highly desirable to modify or supplement the neural mechanisms to prevent or compensate for such disorders. That is the focus of the present invention.
More specifically, the mammalian brain is composed of approximately ten billion nerve cells or xe2x80x9cneuronsxe2x80x9d surrounded by a even greater number of support cells known as neuroglia or astrocyte cells. Neurons, like other cells of the body, are composed of a nucleus, a cytoplasm and a surrounding cell membrane. However, unlike other cells, neurons also possess unique, fiberlike extensions allowing each individual nerve cell to be networked with literally thousands of other nerve cells to establish a neural infrastructure or network. Communication within this intricate network provides the basis for all mental processes undertaken by an organism.
In each nerve cell, incoming signals are received by neural extensions known as xe2x80x9cdendritesxe2x80x9d which may number several thousand per nerve cell. Similarly, neural information is projected along nerve cell xe2x80x9caxonsxe2x80x9d which may branch into as many as 10,000 different nerve endings. Together, these nerve cell axons and dendrites are generally termed xe2x80x9cneuritesxe2x80x9d through which each individual neuron can form a multitude of connections with other neurons. As a result, the number of possible neural connections in a healthy brain is in the trillions, giving rise to tremendous mental capacity. Conversely, when the connections within the neural network break down as nerve cells die or degenerate due to age, disease, oxidative stress, or direct physical insult, the mental capacity of the organism can be severely compromised.
The connection of the individual axons with the dendrites or cell bodies of other neurons takes place at junctions or sites known as xe2x80x9csynapses.xe2x80x9d It is at the synapse that the individual neurons communicate with each other through the flow of chemical messengers across the synaptic junction. The majority of these chemical messengers, or xe2x80x9cneurotransmitters,xe2x80x9d are small peptides, catecholamines or amino acids. When the appropriate stimulus is received by a neural axon connection, the neurotransmitters diffuse across the synapse to the adjacent neuron, thereby conveying the stimulus to the next neuron along the neural network. Based upon the complexity of the information transferred between the nerve cells, it is currently believed that between 50 and 100 distinct neurotransmitters are used to transmit signals in the mammalian brain.
Quite recently, it was discovered that nitric oxide (NO) and carbon monoxide (CO) may function as neurotransmitters. These gaseous molecules appear to participate in a number of neuronal regulatory pathways affecting cell growth and interactions. In the brain, as well as in other parts of the body, CO is produced by the enzyme xe2x80x9cheme oxygenase IIxe2x80x9d (HO). Whether produced from the HO enzyme or from other sources, it is believed that when CO diffuses into a neuron it induces a rise in a secondary transmitter molecule known as xe2x80x9ccyclic guanosine monophosphate xe2x80x9d (cGMP), by modulating an enzyme known as xe2x80x9cguanylate cyclasexe2x80x9d or xe2x80x9cguanylylxe2x80x9d cyclase. Thus, CO acts as a signaling molecule in the guanylyl cyclase regulatory pathway. The resultant increase in cGMP levels appears to modify several neurotropic factors as well as other neuronal factors which may induce, promote or modify a variety of cellular functions including cell growth, protection, and intercellular communication.
Neurotrophic factors are molecules that exert a variety of actions stimulating both the development and differentiation of neurons and the maintenance of cellular integrity and are required for the survival and development of neurons throughout the organism""s life cycle. Generally, neurotrophic factors may be divided into two broad classes: neurotrophins and pleiotrophins. Pleiotrophins differ from the neurotrophins in that they lack a molecular signal sequence characteristic of molecules that are secreted from cells and they also affect many types of cells including neurons. Two effects of neurotrophic factors are particularly important: (i) the prevention of neuronal death and (ii) the stimulation of the outgrowth of neurites (either nascent axons or dendrites). In addition, it appears that CO-induced neurotrophic factors may reduce the membrane potential of nerve cells making it easier for the neurons to receive and transmit signals.
Many of today""s researchers believe that memory is associated with the modification of synaptic activity, wherein the synaptic connections between particular groups of brain neurons become strengthened or facilitated after repeated activation. As a result, these modified connections activate much easier. This type of facilitation is believed to occur throughout the brain but may be particularly prominent in the hippocampus, a brain region which is crucial for memory. The stimulation of neuronal pathways within the hippocampus can produce enhanced synaptic transmission through these pathways for many days following the original stimulation. This process is known as long term potentiation (LTP).
More particularly, long term potentiation is a form of activity-dependent synaptic electrical activity that is exhibited by many neuronal pathways. In this state, generally accepted as a type of cellular memory, nerve cells are more responsive to stimulation. Accordingly, it is widely believed that LTP provides an excellent model for understanding the cellular and molecular basis of synaptic plasticity of the type that underlies learning and memory in vertebrates, including man.
NO and CO are currently the leading candidates for messenger substances that facilitate LTP because inhibitors of these compounds retard the induction of potentiation. The ability to modify neural activity and to increase the ease of LTP using these or other signal transducers could potentially increase learning rates and cognitive powers, possibly compensating for decreased mental acuity. Prior to the present invention, there were no known methods or agents which could operate on the cellular level in vivo to reliably modify cellular and neural regulatory pathways so as to facilitate the LTP of neurons.
In contrast to the enhanced mental capacity provided by long term potentiation, mental functions may be impeded to varying degrees when the neuronal network is disrupted through the death or dysfunction of constituent nerve cells. While the decline in mental abilities is directly related to the disruption of the neural network, it is important to remember that the disruption is occurring on an individual cellular level. At this level the deleterious effects associated with neuronal disruption may be brought about by any one of a number of factors including neurodegenerative diseases and disorders, heart attack, stroke, aging, trauma, and exposure to harmful chemical or environmental agents.
Among the known neurological diseases which adversely impact neuronal function are Alzheimer""s disease and related disorders, Parkinson""s disease, motor neuropathic diseases such as Amyotrophic Lateral Sclerosis, cerebral palsy, multiple sclerosis, and Huntington""s disease. Similar problems may be brought about by loss of neuronal connectivity due to normal aging or through damage to neurons from stroke, heart attack, or other circulatory complications. Direct physical trauma or environmental factors including chemical agents, heavy metals and the like may also provoke neuronal or cellular distress, dysfunction, or death.
Accumulated cellular damage due to oxidative free radicals is believed to be one of the critical factors in a variety of cellular and neurodegenerative diseases including Amyotrophic Lateral Sclerosis, Parkinson""s disease, Alzheimer""s disease, cancer, and aging. Most cells possess a variety of protective mechanisms that guard against cytotoxic free radicals. For example, high levels of glutathione may protect against free radical oxidation. Neurons are deficient in this antioxidant source.
Whatever the cause of the neural disorder or dysfunction, the general inability of damaged nerve cells to undergo substantial regrowth or regeneration under natural conditions has led to the proposal that neurotrophic factors be administered to nerve cells in order to help restore neuronal function by stimulating nerve growth and function. Similarly, stimulating neuritogenesis, or the growth of neurites, by administering neurotrophic factors may contribute to the ability of surviving neurons to form collateral connections and thereby restore neural function.
At present, prior art techniques and compounds have not been effective or practical to directly administer neurotrophic factors to a patient suffering from a neural disorder. In part, this is due to the complex molecular interaction of the neurotrophic factors themselves and to the synergistic regulation of neural cell growth and neuritogenesis. Neurotrophic factors are the result of a long chemical cascade which is exquisitely regulated on the molecular level by an intricate series of transmitters and receptors. Accordingly, neuronal cells are influenced by a concert of different neurotrophic factors, each contributing to different aspects of neuronal development at different times. Neurotrophic factors are, effectively, the tail end of this cascade and thus are one of the most complex components of the regulatory pathway. As such, it was naive for prior art practitioners to assume that the unattenuated administration of single neurotrophic factors at random times (from the cells viewpoint) could substantially improve cell activity or regeneration. In contrast, modification of the regulatory pathway earlier in the cascade could allow the proper growth factors to be produced in the correct relative amounts and introduced into the complex cellular environment at the appropriate time.
Other practical considerations also preclude the prior art use of neurotrophic factors to stimulate the regeneration of the neuronal network. Neurotrophic factors (including neurotrophins and pleiotrophins) are large proteins and, as such, are not amenable to normal routes of medical administration. For example, these proteins cannot be delivered to a patient or subject orally as the patient""s digestive system would digest them before they reached the target neural site. Moreover, due to their relatively large size, the proteins cannot cross the blood brain barrier and access the most important neurological site in the body. Alternatively, the direct injection of neurotrophic factors into the brain or cerebrospinal fluid crudely overcomes this difficulty but is fraught with technical problems of its own which have thus far proven intractable. For example, direct infusion of known neurotrophins into the brain has proven impractical as it requires administration over a period of years to provide therapeutic concentrations. Further, direct injection into the brain has been associated with dangerous swelling and inflammation of the nerve tissue after a very short period of time. Thus, as theoretically desirable as the direct administration of neurotrophic factors to a patient may be, at the present time, it is unfeasible.
Accordingly, it is a general object of the present invention to provide methods and associated compositions and medicaments for effectively modifying mammalian cells, neurons, cellular activity, or neural activity to achieve a variety of beneficial results. These results include protection against oxidative stress and damage by free radicals and more generalized physiological responses such as reductions in mammalian blood pressure.
Thus, it is another object of the present invention to provide methods and associated compositions and medicaments for treating mammalian neurological diseases and cellular disorders.
It is yet another object of the present invention to provide methods and associated compositions and medicaments for inducing long term changes in the membrane potential of a mammalian neuron.
It is still another object of the present invention to provide methods and associated compositions and medicaments for inducing the in vivo physiological production and administration of genetically encoded molecules and neurotrophic factors within cells.
It is a further object of the present invention to provide methods and associated compositions and medicaments for enhancing the neurotogenic effects of neurotrophic factors in a physiological environment.
These and other objects are accomplished by the methods, compositions, and medicaments of the present invention which, in a broad aspect, provide for the selective inducement of the in vivo genetic expression and resultant production of naturally occurring genetically encoded molecules including neurotrophic factors, and for the modification of cellular and neural activity through the treatment of mammalian cells and neurons with at least one carbon monoxide dependent, guanylyl cyclase modulating, purine derivative.
As will be appreciated by those skilled in the art, the in vivo activation or derepression of genetic expression and the exemplary modifications of cellular and neural activity brought about by the methods, compositions, and medicaments of the present invention may be expressed in a variety of forms or combinations thereof. For example, the treatment of a mammalian cell or neuron through the teachings of the present invention may result in the cell""s direct self-administration of the in vivo expressed molecule(s) through the enhanced cellular production of various naturally occurring genetically encoded compounds, such as proteins and neurotrophic factors, or in the stimulation of the activity of those compounds and their subsequent effect on naturally occurring cellular or neuronal metabolism, function, development, and survival. These subsequent effects can include protection from free radical oxidation and cellular destruction, stabilization of cell receptors against other factors, the endogenous production of carbon monoxide and antioxidant compounds, and even reductions in blood pressure via carbon monoxide activated cellular mechanisms. The methods and medicaments of the present invention may also stimulate the growth, development and survival of the cell or neuron directly without the deleterious effects of prior art factor methodology. Further, the present invention may be used to lower or change the membrane potential of the cell, increasing its plasticity and inducing long term potentiation.
Exemplary carbon monoxide dependent guanylyl cyclase modulating purine derivatives useful for practicing the present invention include guanosine, inosine pranobex and 4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid (AIT-082). Unlike prior art compounds, these compounds may be administered directly to a patient either orally or through injection or other conventional routes. These exemplary compounds are nontoxic and will cross the blood-brain barrier as well.
In a further, more specific aspect, the methods and compositions of the present invention may be used for the treatment or prophylactic prevention of neurological diseases and other cellular disorders, including those brought about by disease, oxidative stress, age, trauma or exposure to harmful chemical agents. By promoting the survival, growth and development of individual neurons and cells, the present invention facilitates the regeneration and development of the neural network and alleviates the manifestations of cellular and neural dysfunction.
Of course, those skilled in the art will appreciate that pharmaceutical compositions and medicaments may be formulated incorporating effective concentrations of the carbon monoxide dependent guanylyl cyclase modifying purine derivatives of the present invention along with pharmaceutically acceptable excipients and carriers. These pharmaceutical compositions may be administered orally, transdermally, topically or by injection. Moreover, as the active agents used in the methods of the present invention can cross the blood-brain barrier, they do not have to be injected or infused directly into the brain or central nervous system.
In yet another aspect, the methods and compositions of the present invention may be used to induce long term changes in the membrane potential of a mammalian neuron. These long term potentiation changes may lead to increased membrane plasticity with a corresponding enhancement of cellular memory. In turn, this enhanced cellular memory may elevate the mental capacity of the subject leading to faster learning and increased retention of material.
Other objects, features and advantages of the present invention will be apparent to those skilled in the art from a consideration of the following detailed description of preferred exemplary embodiments thereof taken in con-junction with the data expressed in the associated figures which will first be described briefly.